A prominent goal of synthetic biology is to produce novel biological systems that carry out specified desired functions such as the incorporation of biosynthetic pathways into host cells. As such, synthetic biology requires tools for the selection of genetic components that are inserted or removed from host cells, as well as tools for selective mutation of genetic components within host cells.
One application of synthetic biology is the development of novel isoprenoid synthesis pathways in yeast in order to manufacture isoprenoids at reduced costs relative to conventional techniques. Conventional techniques for manufacturing many isoprenoids, a diverse family of over 40,000 individual compounds, requires their extraction from natural sources such as plants, microbes, and animals. The elucidation of the mevalonate-dependent (MEV) and deoxyxylulose-5-phosphate (DXP) metabolic pathways has made biosynthetic production of some isoprenoids feasible. For instance, microbes have been engineered to overexpress a part of or the entire MEV metabolic pathway for production of an isoprenoid named amorpha-4, II-diene. See U.S. Pat. Nos. 7,172,886 and 7,192,751, which are hereby incorporated by reference.
U.S. Pat. No. 7,659,097 discloses how the activity of the MEV and DXP pathways can be altered in a number of ways in order to increase the synthesis of various isoprenoids. Such alterations include, but are not limited to, expressing a modified form of any respective enzyme in the MEV or DXP pathways so that they exhibit increased solubility in the host cell, expression of an altered form of the respective enzyme that lacks a domain through which the activity of the enzyme is inhibited, expressing a modified form of the respective enzyme that has a higher Kcat or a lower Km for its substrate, or expressing an altered form of the respective enzyme that is not affected by feed-back or feed-forward regulation by another molecule in the pathway. Moreover, the nucleic acid sequences encoding the enzymes in such synthetic pathways can be modified to reflect the codon preference of the host cell in order to effect a higher expression of such enzymes in the host cell. Further still, multiple copies of enzymes in such biosynthetic pathways may be introduced into host cells to effect a higher expression of such enzymes. Further still, such enzymes may be placed under the control of powerful promoters in the host cell to effect a higher expression of such enzymes. See U.S. Pat. No. 7,569,097, which is hereby incorporated by reference. The above illustrates just some of the many changes to the locus of a host cell or organism that are made in order to realize a synthetic biology design goal such as the manufacture of isoprenoids.
As the above illustrates, the realization of synthetic biology goals is best achieved through an iterative trial and error approach in which tens, hundreds, or even thousands of different design attempts are tested in vivo in a host cell or organism on a periodic basic (e.g., daily, weekly, monthly) to determine if a design goal has been reached and to improve upon such design goals. As such, it is clear that what is needed in the art are improved platforms for realizing such design goals faster, more efficiently, and in an even more economical fashion.
One facet of a platform for realizing synthetic biology design goals is mechanisms for reducing design goals into a form that is interpretable by a compiler. In one approach, Pedersen and Phillips, 2009, “Towards programming languages for genetic engineering of living cells”, J. R. Soc. Interface 6, S437-S450 provide a formal language for genetic engineering of living cells (GEC) in which one or more in silico databases of parts are searched by a compiler for a set of parts that satisfy a design goal. See also, U.S. Patent Application Publication No. 2011/0054654 in which GEC is also described. The work of Pedersen and coworkers provides a satisfactory framework for modeling complex pathways in silico. Such in silico models can then be used to make in silico predictions on what changes to the model would achieve a desired design goal. However, the data that would make such in silico modeling more useful, such as the molecular properties of a number of components of molecular pathways under a number of different reaction conditions, is presently unavailable. Consequently, to date, the work of Pederson and coworkers has not eliminated the need for an iterative trial and error approach to realizing a synthetic biology design goal in which tens, hundreds, or even thousands of different design attempts are tested in vivo on a periodic basic (e.g., daily, weekly, monthly).
The drawbacks of iterative trial and error approaches are the time and costs that such approaches take. It takes extensive resources, including time and money, to make all the constructs necessary for a design attempt and to test the design attempt in vivo. For each design attempt, the constructs, termed engineered nucleic acid constructs, which effect the desired changes to the locus of a host cell or organism, need to be made. This often requires the custom synthesis of oligonucleotide primers in order to subclone desired nucleic acid components from a genomic library and/or to effect desired mutations in existing nucleic acid sequences. Such engineered nucleic acid constructs are then introduced into a host cell or organism where they either recombine with a locus of the host genome or exist in a stable vector form. As such, the design of even a limited number of engineered nucleic acid constructs may require the synthesis of dozens or even hundreds of custom oligonucleotide primers in order to make the needed engineered nucleic acid constructs using existing template nucleic acids, such as existing constructs or nucleic acids in a genomic library.
Thus, despite advances in the field of synthetic biology, there remains a need for improved systems, compositions, and methods that provide for the rapid and ordered assembly of nucleic acid components into engineered nucleic acid constructs. Particularly needed are systems and methods that reduce the cost and increase the speed of the iterative trial and error approach that is used in synthetic biology applications, including the construction of engineered nucleic acid constructs. These and other needs are met by systems, compositions, and methods of the present disclosure.